In many fields of life sciences research, including biological, biomedical, genetic, fermentation, aquaculture, agricultural, forensic and environmental research, there is a need to identify nucleic acids, as well as quantify those nucleic acids, in pure solutions and in biological samples. Such applications require a fast, sensitive, and selective methodology that can detect minute amounts of nucleic acids in a variety of media, whether or not the nucleic acid is contained in cells.
The detection of nucleic acids by fluorescent dyes such as SYBR Green I (SG) has been applied successfully in the detection of nucleic acids in gels, in solution, in the determination of DNase or telomerase activities, in fluorescence imaging techniques, in flow cymetry, in real-time PCR, in biochip applications, and in the quantification of double stranded DNA, e.g., in crude extracts.
SG was shown to intercalate at low dye/base pair ratios (dbprs) (Zipper et al., Nucl. Acids Res., 32:e103 (2004)). In agreement with most simple intercalators and the fluorescent DNA binding dye PicoGreen (PG), SG did not display a marked sequence binding preference at low dbprs, although a significant sequence dependence of SG binding was observed at high dbprs (Zipper et al.). PG was not sequence specific at low dbprs, however, at high dbprs, which are generally used for quantification of DNA in solution, PG had some sequence specificity (Zipper et al.).
Zipper et al. disclose that dbprs>0.2 should be applied to discriminate between single stranded DNA and double stranded DNA, and that for maximum double stranded DNA selectively, for instance, with respect to other compounds such as RNA, proteins, and the like, dbprs of at least 10 should be employed. It is also disclosed that the use of higher dbprs can reduce the impact of salts, quenchers and dsDNA sequence specificity, which can affect SG real-time PCR and melting curve analyses (Zipper et al.). Zipper et al. caution that inhibition of PCR as well as the potential degradation of dsDNA may occur at high dbprs and that in PCR the dbpr is not constant.
EvaGreen® (EG) is a DNA binding dye with a lower binding affinity for both double stranded DNA and single stranded DNA than SG (Mao et al., BMC Biotech., 7:76 (2007)). Mao et al. disclose that EG showed no apparent preference for either GC- or AT-rich sequences while SG had a slight preference for AT-rich sequences. Both dyes showed substantially lower affinity towards single stranded DNA than toward double stranded DNA, however, both EG and SG exhibited PCR interference when used at high dye concentrations, as evident by the delayed Ct and/or nonspecific product formation (Mao et al.). Mao et al. disclose that the problem worsened when the chain extension time was shortened or when the amplicon size was relatively long (>500 bp). The differences in qPCR performance between the two dyes was attributed to their differences in DNA binding profiles (Mao et al.).